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Background: In females, one X-chromosome is transcriptionally silenced by methylation of CpG sites in every cell during early embryogenesis. The methyl groups needed for DNA methylation are provided by the methylenetetrahydrofolate reductase (MTHFR). A polymorphism in the enzyme (677C>T, rs1801133) affects MTHFR concentrations and possibly also methylation of X-chromosomal genes. Whether X-chromosome inactivation (XCI) by methylation is associated with the MTHFR 677C>T genotype is currently unknown. It is also unclear whether the imprinting subsequently influences factor VIII activity in carrier females.
Methods: We determined XCI in 61 hemophilia A carrier females and 174 control females by analysis of the human androgen receptor locus. Genotyping of MTHFR 677C>T was performed by mutagenically separated PCR, FVIII:C was tested by one-stage coagulation assays.
Results: Slight skewing of XCI (X1:X2<1:3) was not associated with FVIII:C activity in hemophilia A carriers and controls. In contrast, strongly skewed XCI, which was observed in 10 carrier females, was associated with FVIII:C activity levels. If the X-chromosome carrying the mutated F8 gene was methylated, FVIII:C levels were higher. In contrast, FVIII:C levels were lower if the X-chromosome with the intact F8 gene was imprinted. We found a significant association of the 677TT MTHFR genotype with low FVIII in carrier females. The MTHFR genotype was not associated with FVIII:C in control females.
Conclusion: Highly skewed imprinting of the X-chromosome influenced FVIII:C levels in carriers of hemophilia A. We found a significant association of MTHFR 677TT with low FVIII: C in hemophilia A carriers. Surprisingly, the MTHFR 677TT genotype was not significantly associated with X-chromosome inactivation. To elucidate the underlying mechanisms further studies will be necessary.